Method for Producing Natively Folded Proteins in a Prokaryotic Host

a prokaryotic host and native folding technology, applied in the field of native folding proteins in prokaryotic hosts, can solve the problems of difficult biotech industry production of proteins containing disulfide bonds on a large scale, the rate-limiting step of native disulfide bond formation, and the confusion of their precise individual roles

Active Publication Date: 2012-08-09
UNIV OF OULU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0076]The advantage of the present invention is that the protein of interest is formed in soluble form. Typically no denaturation and renaturation steps of the protein are needed. Furthermore, the protein is produced directly in biologically active form.
[0077]Commercially significant proteins which may be produced by using the present invention comprise for example insulin, blood coagulation factors, cytokines, chemokines, interferons, growth hormones and single chain antibodies.
[0078]In this disclosure as examples of such proteins are the luminal domain of human tissue factor, E. coli alkaline phosphatase and phytase, bovine pancreatic trypsin inhibitor (BPTI), human colony stimulating factor 3 (CSF3), bone morphogenic protein 4 (BMP4), tissue plasminogen activator (t-PA), interferon α2, interleukin 6, interleukin 17, resistin and growth hormone 1.
[0079]In this disclosure the ability to generate “soluble protein” or “insoluble inclusion bodies” is deduced from SDS-PAGE analysis of total and soluble fractions of a cell lysate. The generation of insoluble inclusion bodies is a common occurrence when proteins that natively contain d...

Problems solved by technology

Hence, it is unsurprising that native disulfide bond formation is often the rate-limiting step in the folding of proteins in vitro and in vivo.
However, while some of the participants in the cellular process are known, their precise individual roles are still largely confused.
Currently proteins that contain disulfide bonds are difficult for the biotech industry to produce on a large scale.
Due to this the recombinant proteins are unable to attain their native conformation and form insoluble inclusion bodies.
However, it is costly, complex and generally inefficient.
While native E. coli disulfide bond containing proteins fold efficiently in t...

Method used

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  • Method for Producing Natively Folded Proteins in a Prokaryotic Host
  • Method for Producing Natively Folded Proteins in a Prokaryotic Host
  • Method for Producing Natively Folded Proteins in a Prokaryotic Host

Examples

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example 1

[0157]Plasmid constructs used in the protein expressions were generated as part of prestudies (see FIG. 5). The original constructs were polycistronic with multiple genes encoded by a single mRNA driven by a T7 promoter system (IPTG inducible) in a modified version of pET23. These included the protein of interest alone (A), this plus the sulfhydryl oxidase (B) or plus the sulfhydryl oxidase and either PDI or DsbC as a thiol-disulfide isomerase (C).

[0158]Subsequent constructs had the sulfhydryl oxidase (D) or the sulfhydryl oxidase plus the thiol-disulfide isomerase (E) on an arabinose promoter as part of a modified version of pLysS. Constructs D and E are fully compatible with construct A and allow easy inter-conversion of the protein of interest in the system. In addition, constructs D and E allow pre-expression or co-expression of the sulfhydryl oxidase, or the sulfhydryl oxidase and thiol-disulfide isomerase, and the protein of interest.

example 2

Efficient Production of the Luminal Domain of Human Tissue Factor

[0159]Tissue factor (TF), also known as thromboplastin factor III, is a protein involved in the coagulation of blood. It is a transmembrane protein whose luminal domain contains two sequential disulfide bonds.

[0160]pVD81, is a derivative of pET23a which has cloned into the multi-cloning site a gene encoding for an N-terminal hexa-histidine tag (MHHHHHHM) (SEQ ID NO: 11) followed in frame with the luminal domain of human tissue factor (sTF) as represented by the fragment Ser 33-Glu 251 of the full length protein. This vector expresses sTF upon induction with IPTG.

[0161]pVD77 is a derivative of pVD81, in which the gene for the sulfhydryl oxidase Erv1p from S. cerevisiae has been cloned (Met 1-Glu 189) (SEQ ID NO: 5) after the gene for sTF (with suitable ribosome binding sites to initiate translation of both; see FIG. 5). This polycistronic vector co-expresses sTF and Erv1p upon induction with IPTG.

[0162]E. coli strains t...

example 3

Efficient Production of E. coli Alkaline Phosphatase

[0165]Alkaline phosphatase is a hydrolase which removes phosphate groups from many types of molecules. The bacterial enzyme folds in the periplasm. It has two sequential disulfide bonds whose formation is essential for activity. Since it is easily assayed bacterial alkaline phosphatase is widely used as a model protein for disulfide bond formation in vivo.

[0166]pVD80, is a derivative of pET23a which has cloned into the multi-cloning site a gene encoding for an N-terminal hexa-histidine tag (MHHHHHHM) (SEQ ID NO:12) followed in frame with the mature form of E. coli alkaline phosphatase (PhoA) as represented by the fragment Arg 22-Lys 471 of the full length protein. This vector expresses alkaline phosphatase upon induction with IPTG.

[0167]pVD82 is a derivative of pVD80, in which the gene for the sulfhydryl oxidase Erv1p from S. cerevisiae has been cloned (Met 1-Glu 189) (SEQ ID NO: 5) after the gene for E. coli alkaline phosphatase (...

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Abstract

The present invention relates to a method for producing a protein of interest containing one or more disulfide bonds in its native state. The method comprises that a prokaryotic host cell is genetically engineered to express the protein of interest and a sulfhydryl oxidase in the cytoplasm of the host cell. The protein of interest is formed in a soluble form and contains disulfide bonds due to the presence of the sulfhydryl oxidase in the cytoplasm of said host cell. The present invention relates also to a prokaryotic host cell and a vector system for producing a protein of interest containing natively folded disulfide bonds.

Description

PRIORITY CLAIM[0001]This is a continuation-in-part application of International application number PCT / FI2010 / 05448 filed on Jun. 2, 2010 claiming priority of the Finnish national patent application number 20095615 filed on Jun. 2, 2009, the contents of both of which are incorporated herein by reference in their entirety.SEQUENCE DATA[0002]This application contains sequence data provided in computer readable form and as PDF-format. The PDF-version of the sequence data is identical to the computer readable format.FIELD OF THE INVENTION[0003]This invention relates to a method, a host cell and a vector system for producing a protein of interest containing one or more disulfide bonds in its native state. In particular, the invention relates to a method, a host cell and a vector system for producing such proteins in a prokaryotic host.DESCRIPTION OF RELATED ART[0004]Many proteins and enzymes of biotechnological importance contain structure stabilizing disulfide bonds, with an estimated o...

Claims

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Application Information

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IPC IPC(8): C12P21/06C12N15/63C12N1/21
CPCC12N9/0051C12N9/90C12Y503/04001C12P21/02C12Y108/03002C12N15/70C07K14/4703C07K14/51C07K14/53C07K14/54C07K14/5412C07K14/555C07K14/575C07K14/61C07K14/70596C12P21/00
Inventor RUDDOCK, LLOYD
Owner UNIV OF OULU
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